Method for the isomerisation of glucose

Abstract

Disclosed is a method for the isomerization of glucose by reduction to sorbitol and subsequent oxidation to fructose, in which the redox cofactors NAD.sup.+/NADH and NADP.sup.+/NADPH are regenerated in a one-pot-reaction, wherein one of the two redox cofactors is obtained in the reduced form thereof and the other redox cofactor in the oxidized form thereof as a result of at least two additional enzymatically catalyzed redox reactions (product forming reactions) taking place in the same reaction batch, wherein a) in the regeneration reaction, which transfers the reduced cofactor back to its originally oxidized form, oxygen or a compound of the general formula R.sub.1C(O)COOH is reduced, and b) in the regeneration reaction, which transfers the oxidized cofactor back to its originally reduced form, a compound of the general formula R.sub.2CH(OH)R.sub.3 is oxidized, wherein R.sub.1, R.sub.2 and R.sub.3 have different meanings in the compounds, characterized in that a mixture of glucose and fructose is used as a starting material. Furthermore, the use of fructose thus produced in a method for producing furan derivatives is disclosed.

Claims

1. A method for the production of D-fructose, comprising: providing a mixture comprising D-glucose and D-fructose as a starting material; isomerising D-glucose to D-fructose by reduction of D-glucose to D-sorbitol and subsequent oxidation of D-sorbitol to D-fructose using redox cofactors NAD.sup.+/NADH and NADP.sup.+/NADPH, wherein one of the redox cofactors is obtained in a reduced form and the other redox cofactor is obtained in an oxidised form as a result of at least two enzymatically catalysed redox reactions (product forming reactions) taking place in a one-pot reaction; and in a regeneration reaction, regenerating the redox cofactors in the one-pot reaction, wherein a) in the regeneration reaction, which transfers the reduced cofactor back to its originally oxidised form, oxygen or a compound of the general formula ##STR00007## wherein R.sub.1 is a straight or branched (C.sub.1-4)-alkyl group or a (C.sub.1-4)-carboxyalkyl group, is reduced, and b) in the regeneration reaction, which transfers the oxidised cofactor back to its originally reduced form, a (C.sub.4-8)cycloalkanol or a compound of the general formula ##STR00008## wherein R.sub.2 and R.sub.3 are each independently selected from the group consisting of H, (C.sub.1-6)alkyl, wherein the alkyl is straight or branched, (C.sub.2-6)alkenyl, wherein the alkenyl is straight or branched and contains one to three double bonds, aryl, carboxyl, or (C.sub.1-4)carboxyalkyl, is oxidised.

2. The method according to claim 1, wherein in a) a compound of the general formula I, wherein R.sub.1 is a substituted or unsubstituted (C.sub.1-4)alkyl group, is reduced, and in b) a compound of the general formula II, wherein R.sub.2 and R.sub.3 are independently selected from the group consisting of H, (C.sub.1-6)alkyl, wherein alkyl is straight or branched, (C.sub.2-6)alkenyl, wherein alkenyl is straight or branched and optionally contains up to three double bonds, cycloalkyl, aryl, (C.sub.1-4)carboxyalkyl, if compound I is a pyruvate, optionally also carboxyl, is oxidised.

3. The method according to claim 1, wherein in b) a compound of formula II, wherein R.sub.2 and R.sub.3 are independently selected from the group consisting of H, (C.sub.1-C.sub.6)alkyl, wherein alkyl is straight or branched, (C.sub.2-6)alkenyl, wherein alkenyl is straight or branched and contains one to three double bonds, aryl, carboxyl, or (C.sub.1-4)carboxyalkyl, is oxidised.

4. The method according to claim 1, wherein the isomerisation of D-glucose to D-fructose follows the following reaction scheme 1: ##STR00009##

5. The method according to claim 1, wherein the oxidation reaction(s) and reduction reaction(s) take place parallel in time.

6. The method according to claim 1, wherein the oxidation reaction(s) and reduction reaction(s) do not take place parallel in time.

7. The method according to claim 1, wherein in the regeneration reaction, which transfers the oxidised cofactor NAD(P).sup.+ back to its originally reduced form NAD(P)H, 2-propanol is oxidised to acetone by means of an alcohol dehydrogenase.

8. The method according to claim 1, wherein in the regeneration reaction, which transfers the reduced cofactor NAD(P)H back to its originally oxidised form NAD(P).sup.+, oxygen is reduced to water by means of an NADH oxidase.

9. The method according to claim 1, wherein the substrate(s) for the oxidation reaction(s) involved in the product formation is/are present in the one-pot reaction in a concentration of 5% (w/v) or more.

10. The method according to claim 1 wherein the fructose obtained according to claim 1 is isolated in a crystallised form.

11. The method according to claim 1, wherein at least one of R.sub.2 or R.sub.3 is selected from the group consisting of (C.sub.6-12)aryl, (C.sub.1-4)carboxyalkyl, and (C.sub.3-8)cycloalkyl.

12. The method according to claim 1, wherein the substrate(s) for the oxidation reaction(s) involved in the product formation is/are present in the one-pot reaction in a concentration of 7% (w/v).

13. The method according to claim 1, wherein the substrate(s) for the oxidation reaction(s) involved in the product formation is/are present in the one-pot reaction in a concentration of 9% (w/v).

14. A method for obtaining furan derivatives from a mixture of glucose and fructose, comprising: A) converting a mixture comprising D-glucose and D-fructose to D-fructose in an enzymatic method by use and regeneration of redox cofactors, wherein one of two redox cofactors is obtained in a reduced form and the other redox cofactor is obtained in an oxidised form as a result of at least two additional enzymatically catalysed redox reactions taking place in a one-pot reaction, wherein D-glucose is converted to D-fructose with the involvement of two or more oxidoreductases, and B) converting the D-fructose obtained in A) to furan derivatives.

15. The method according to claim 14, wherein in stage B) an acidic catalyst and a solvent are used.

16. The method according to claim 14, wherein the conversion of D-fructose to furan derivatives in stage B) is carried out as either a batch process or as a continuous process.

17. The method of claim 16, wherein the conversion of D-fructose to furan derivatives in stage B) is carried out under microwave heating.

18. The method according to claim 15, wherein during conversion of D-fructose to furan derivatives in stage B) the acidic catalyst used is a homogeneous acidic catalyst; a heterogeneous acidic catalyst a Lewis acid catalyst, a SILP catalyst.

19. The method according to claim 18, wherein the homogeneous acidic catalyst comprises at least one of sulphuric acid or hydrochloric acid.

20. The method according to claim 18, wherein the heterogeneous acidic catalyst comprises an ion exchanger.

21. The method according to claim 18, wherein the Lewis acid catalyst comprises at least one of CrCl.sub.2, AlCl.sub.3 or SiO.sub.2MgCl.sub.2.

22. The method according to claim 14, wherein the furan derivative is hydroxyl methyl furfural of the following formula ##STR00010##

Description

EXAMPLE 1

Production of Fructose from Glucose-Fructose Syrup by Glucose Isomerase Followed by a Two-Stage Redox Process

(1) 750 mg of D-glucose were dissolved in 50 mM of Tris buffer (pH=8.0 at 25 C.) to a total volume of 5 ml. To this mixture, 250 mg of immobilised glucose isomerase from Streptomyces murinus (Sigma-Aldrich, Novozymes Sweetzyme ITC)) were added, and the suspension was gently shaken at 50 C. for 6 h. This led to the conversion of approximately 33% of glucose to fructose. The glucose isomerase was removed by centrifugation (5000 g, 1 min). In a 2 ml glass vessel, 400 l of the solution were then treated with 10 l of Tris HCl (0.5 M, pH=8.0), 20 l of xylose reductase from Candida tropicalis (overexpressed in E. coli BL21 (DE3), 280 U/ml), 30 l of alcohol dehydrogenase from Lactobacillus kefir (overexpressed in E. coli BL21 (DE3), 130 U/ml), and 35 l of 2-propanol. The reaction was carried out in an open system in which the glass vessel was shaken for 24 h at 40 C. (Eppendorf Thermomix, 850 rpm). The open system allows the reaction product of acetone to evaporate, which drives the reaction towards sorbitol formation. The following supplementary additions were made: 15 l of 2-propanol after 4 h, 25 l of 2-propanol after 18 h, and 50 l of water after 18 h. 98.5% of the glucose still present were converted to sorbitol. The mixture contained a total of approximately 71% of sorbitol, 28% of fructose, and 1% of glucose. In a further reaction step, 60 l of NADH oxidase from Leuconostoc mesenteroides (overexpressed in E. coli BL21 (DE3), 350 U/ml) and 40 l of sorbitol dehydrogenase from Bacillus subtilis (overexpressed in E. coli BL21 (DE3), 50 U/ml) were added. Again, the reaction took place in an open system in order to guarantee oxygen supply to the NADH oxidase reaction. The reaction vessel was shaken for 48 h at 25 C. (Eppendorf Thermomix, 850 rpm). A mixture of 60% of D-fructose, 35.2% of D-sorbitol, and 4.7% of D-glucose was obtained.

EXAMPLE 2

Materials and Methods for the Conversion of D-Fructose to Furan Derivatives

(2) Dehydration reactions of D-fructose to HMF were carried out under different reaction conditions, optionally as standard batch process under microwave-assisted heating or by continuous-flow conditions. Surprisingly, it was found that compared to known systems the use of NMP as solvent in the reaction in combination with homogeneous or heterogeneous catalysts results in higher yields, in the microwave-assisted method as well as under continuous-flow conditions.

(3) Synthesis of SiO.sub.2MgCl.sub.2

(4) SiO.sub.2MgCl.sub.2 was produced similarly to the protocol according to Yasuda et al. (Yasuda, M.; Nakamura, Y.; Matsumoto, J.; Yokoi, H. Shiragami, T. Bull. Chem. Soc. Jpn. 2011, 84, 416-418).

(5) Synthesis of SILPs

(6) The SILP catalyst was produced according to known protocols (Fu, S.-K.; Liu, S.-T. Synth. Commun 2006, 36, 2059-2067) using N-methyl-imidazol. For immobilisation, the ionic liquid obtained was mixed with 200 wt % of silica gel in dry chloroform (100 ml per 10 g SiO.sub.2) and heated for 24 h to 70 C. The solid obtained was filtered off, washed with chloroform, and dried under reduced pressure. The silica gel obtained had a catalyst load of approximately 16 wt %.

(7) General Conditions of Batch Reactions

(8) If not stated otherwise, all batch reactions were carried out in a 4 ml screw-lid glass jar. Heating was carried out in suitable aluminium blocks to the desired temperature.

(9) Microwave Reactions in the Batch Process

(10) Microwave reactions in a batch process were carried out in a Biotage Initiator Sixty laboratory microwave equipped with an autos ampler in order to allow sequential reactions. The absorption level was set to the maximum value, which automatically controls the maximum energy input at 400 W.

(11) Stopped-Flow Microwave Reactions and Continuous-Flow Reactions

(12) Stopped-flow reactions for optimising semi-continuous processes were carried out on a CEM Discover System with CEM Voyager Upgrade and via an external pressure sensor. For reactions in continuous processes, a cartridge-based reactor system X-Cube by ThalesNano, equipped with a Gilson GX-271 autosampler for automatic product collection, was used. Here, two quartz sand cartridges (CatCart, 704 mm) were incorporated as reactions zones.

(13) Alternatively, a perfluoro alkoxy alkane capillary was used (PFA capillary, 0.8 mm inner diameter, 1.6 mm outer diameter), which was wound around a heatable aluminium cylinder. The substrates were added via a Shimadzu LC-10AD HPLC pump at the desired flow rate. Exact volumes (column 16.0 ml; dead volume before and after the column 1.0 ml each) were determined by monitoring defined flow rates of the pure solvent by means of a digital stop watch.

(14) Analysis of the Reactions for Converting D-Fructose to Furan Derivatives

(15) For a quantitative HPLC analysis, reaction samples (22 l, if not stated otherwise) were diluted with deionised water to 1 ml. For reaction samples having different concentrations, dilution was adapted so that the maximum concentration did not exceed 2 mg/ml.

(16) To this solution, 100 L of 3-hydroxy benzyl alcohol were added as internal standard, followed by thorough mixing of the sample. Solid residues were separated by centrifugation (5 min, 20000 G) or filtration (Phenex PTFE, 4 mm, 0.2 m). Quantification was based on the areas of the peaks in the RI spectrum compared to the internal standard.

(17) The samples were analysed via HPLC on a Thermo Scientific Surveyor Plus System or a Shimadzu Nexera System, each equipped with a PDA Plus and RI detector. For separation, the stationary phase was an ion exchange column by Phenomenex (Rezex RHM-Monosaccharide H+ (8%), 1507.8 mm, built of a crosslinked matrix of sulfonated styrene and divinyl benzene, FE form), and the eluent consisted of water (HPLC grade) and 0.1% TFA (HPLC grade). The column temperature was kept constant at 85 C., optimising running time to 25 min Product quantification was carried out by means of an internal standard via integration of the RI signal. In addition, the wavelengths of 200 nm, 254 nm and 280 nm were recorded by PDA for further reaction analysis.

(18) GP1D-Fructose Dehydration in the Batch Process

(19) In a standard reaction for reaction optimisation, 100 mg of D-fructose (0.56 mmol) and a desired amount of the respective catalyst were put into a glass vial and treated with 1 ml freshly distilled NMP. The solution/suspension obtained was heated to the selected temperature and allowed to react for the desired time.

(20) GP2D-Fructose Dehydration in the Microwave Batch Process

(21) In a standard reaction for reaction optimisation, 100 mg of D-fructose (0.56 mmol) and the desired amount of the respective catalyst were added to a microwave vessel (0.5-2.0 ml). The vessel was equipped with a magnetic stirring bar, and 1 ml of NMP was added. The radiation intensity of the microwave was automatically set by a company-internal regulation algorithm in order to achieve the desired temperature. Quick cooling of the reaction vessel was achieved by blowing in pressurised air of at least 6 bar.

(22) GP3D-Fructose Dehydration in the Microwave Stopped-Flow Process

(23) In a standard reaction for reaction optimisation, a D-fructose standard solution (1 ml; c=100 mg/ml in NMP) and hydrochloric acid (100 l; c=1 mol/l) were added to a microwave vessel equipped with a magnetic stirring bar. After sealing the vial with a snap cap, the solution was heated for the desired time to the desired temperature. In order to achieve the fastest possible heating, the energy applied was set according to the following Table 1.

(24) TABLE-US-00002 TABLE 1 Power settings of the microwave and associated temperatures Temperature Power setting 100 C. 50 W 125 C. 65 W 150 C. 100 W 180 C. 125 W 200 C. 140 W 220 C. 160 W

(25) Quick cooling of the reaction vessel was achieved by blowing in pressurised air of at least 6 bar.

(26) GP4D-Fructose Dehydration in the Cartridge-Based Reactor System

(27) In a standard reaction for reaction optimisation, a D-fructose standard solution (1 ml; c=100 mg/ml in NMP) was mixed with hydrochloric acid (c=1 mol/l) and pumped into the reaction system by a reagent pump. During the heating process, several preliminary samples were taken in order to monitor a stable temperature and a stable flow rate. The reaction temperatures selected were 150 C., 180 C. and 200 C., while the reaction pressure was regulated at 40 bar. Flow rates between 0.2 and 0.6 ml/min were selected. Reaction samples were taken at amounts of 2.5 ml and analysed.

EXAMPLE 3

Use of Sulphuric Acid as Catalyst for Dehydrating D-Fructose

(28) Different temperatures, reaction times and acid concentrations were compared. The reaction was carried out according to GP1 (Example 4). The catalyst used was either 100 l of 1N sulphuric acid or 10 l of concentrated sulphuric acid. The results are summarised in Table 1.

(29) TABLE-US-00003 TABLE 1 Sulphuric acid as catalyst for dehydrating D-fructose Fructose Tem- Reaction con- HMF HMF LS Catalyst perature time sumption yield selectivity yield 1N H.sub.2SO.sub.4 100 C. 3 h 69% 45% 65% <1% 1N H.sub.2SO.sub.4 120 C. 4 h 95% 77% 81% <1% 1N H.sub.2SO.sub.4 150 C. 15 min 98% 88% 90% <1% 1N H.sub.2SO.sub.4 180 C. 10 min 100% 85% 85% <1% H.sub.2SO.sub.4 conc. 120 C. 45 min 98% 85% 90% <1% H.sub.2SO.sub.4 conc. 150 C. 10 min 100% 90% 90% <1% H.sub.2SO.sub.4 conc. 180 C. 5 min 100% 82% 82% <1%

(30) No formation of black, insoluble polymers and humans was observed under the optimum conditions used.

EXAMPLE 4

Use of Sulphuric Acid for Catalysing the Conversion of D-Fructose to Furan Derivatives (Continuous Process)

(31) D-fructose (10% w/v) and concentrated sulphuric acid (1% v/v) were dissolved in N-methyl-2-pyrrolidone. The mixture was pumped through the reactor by means of a PFA capillary with continuous flow (reaction temperature 150 C.). After the first 18 ml had been discarded, further 10 ml were collected for analysis. With various flow rates, the effect of different dwell times in the reactor were tested (Table 10).

(32) TABLE-US-00004 TABLE 10 Sulphuric acid for catalysing the conversion of D- fructose to furan derivatives (continuous process) Flow rate Fructose HMF HMF LS (ml/min) Dwell time consumption yield selectivity yield 0.8 ml/min 20 min 100% 74% 74% <1% 1.6 ml/min 10 min 100% 75% 75% <1% 3.2 ml/min 5 min 100% 76% 76% <1%

(33) No formation of black, insoluble polymers and humins was observed under the conditions analysed.

EXAMPLE 5

Use of Amberlite 15 as Catalyst for Dehydrating D-Fructose

(34) This example shows the use of a strong ion exchanger with sulfonic acid residues based on a macro-crosslinked resin. 100 mg of D-fructose were incubated in the presence of 1 ml of N-methyl-2-pyrrolidone for 3 h at 100 C. under stirring (protocol GP1, Example 2). Amberlite 15 was added as catalyst. Table 2 shows the result of this experiment. A high yield was achieved at the relatively low temperature. The formation of tar-like compounds was avoided.

(35) TABLE-US-00005 TABLE 2 Amberlite 15 as catalyst for dehydrating D-fructose Amount Reaction Fructose HMF HMF LS of catalyst Temp. time consumption yield selectivity yield 10 mg 100 C. 3 h 70% 50% 71% <1%